3D Platform /Ex Vivo Assays for Pharmacology Factsheet Capture the reality of tumor biology by evaluating your compounds in CrownBio’s extensive platform of 3D ex vivo and in vitro assays

Around 95% of new anticancer drugs eventually fail in clinical trials(1), despite robust indications of activity in existing in vitro preclinical models, suggesting that innovative models are required that better mimic tumor biology . To in- crease the speed and efficiency of the preclinical oncology research process a high proportion of drug discovery is current- ly performed using cell cultures. However, culturing cells on flat plasticware results in artificial 2D monolayering, with cells suffering additional stress as they spread and flatten on the plastic surface of the flask and fail to engage in efficient cell-cell and cell-matrix contacts. All of these factors are known to influence gene expression and protein function therefore raising the question as to whether a 2D cell culture model represents, with sufficient fidelity, the corresponding tumorin situ.

Cells that make up tissues follow complex and dynamic 3D arrangements, Our models provide a rich resource for the derivation of primary cells and which are important for their physiology. The 3D architecture of a cell in- cell lines across a range of cancer types with many molecular targets. fluences its ability to respond to external stimuli and activate the appro- priate response. Cancer cells (much like their normal counterparts) ben- Our ex vivo models include freshly isolated cells from PDX tumors (utilized efit from such 3D interactions with each other and with the surrounding in assays immediately on the day of isolation). We have currently validated extracellular matrix by establishing a unique growing niche, the tumor almost 70 PDX-derived primary cells for 3D cell culture assays, with cells de- microenvironment (TME). In order to investigate the efficacy of a new rived from both solid tumors and blood cancer models (detailed in Table 2). compound as an anticancer agent it is necessary to take in to consider- ation the 3D nature of a tumor and its communications with the TME. Table 2: PDX-Derived Primary Cells Validated in 3D Cell Culture 3D cell cultures represent a significant step forward towards capturing Assays the realities of tumor biology in situ. In a 3D cell culture, tumor cells are Models in orange are newly validated. grown to avoid contact with plastic allowing them to keep their native ar- chitecture and to establish interactions with each other, which minimizes Tissue of HuPrime Identifier stress and an artificial response. A comparison between 2D culturing and Origin the improvement presented by 3D cell culture is shown in Table 1. ALL AL5511, AL7443, AL7473 Table 1: Comparison of 2D and 3D Cell Culture AML AM5512, AM7440, AM7577 Brain BN2276, BN2287, BN2331, BN2338 2D 3D Breast BR1282, BR1283, BR1458, BR1474 Cell Shape Flat. Thickness: 3µm Ellipsoid. Thickness: 10-30µm Colorectum CR0029, CR1520, CR1560, CR1574, CR2161, CR2506, CR2518, Environment About 50% of cell surface in Nearly 100% of surface CR2520, CR2524, CR2545, CR3085 contact with culture flask and exposed to other cells or to very small proportion of cell in the matrix Esophageal ES0195, ES0201 contact with neighboring cells Gallbladder GL0440 Cell Differences in differentiation(2), drug metabolism(3), gene express- Behavior ion(4), proliferation rates(5), response to stimuli(6), and viability(7) Liver LI0050, LI0574, LI0612, LI0752, LI0941, LI1037, LI1098, LI1646 Lung LU0038, LU0299, LU0330, LU0367, LU0377, LU0387, LU0395, LU0743, LU0755, LU0884, LU1143, LU1144, LU1147, LU1155, LU1215, LU1271, CrownBio 3D Cell Culture Resources LU1380, LU1423, LU1868, LU1901, LU2503, LU2505, LU2512, LU3075, LU6409 At CrownBio, we offer a variety of ex vivo and in vitro 3D assay systems for Ovarian OV0205, OV0273, OV1658 a range of ex vivo and in vitro models for preclinical oncology research. Pancreas PA1168 Our ex vivo models are derived from HuPrime® and HuKemia® - Stomach GA0037, GA0087, GA0103, GA0114 CrownBio’s collection of highly characterized patient-derived xeno- graft (PDX) models, the largest commercial PDX collection available.

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We also provide fresh frozen PDX cells for 3D assay. Cell suspensions HuPrime models have also been used to establish our propri- are isolated, frozen, and thawed as needed for assays. This can short- etary PrimePanel™ cell line collection(8), consisting of mouse en assay timelines to two weeks, with no lag time while PDX tumor stromal-cell-depleted cancer cell cultures. Our cell lines are donors grow out. CrownBio provides fresh frozen PDX cells from a all early passage (<10) and maintain essential histopathological fea- range of cancer types (shown in Table 3), with further models under tures and genetic profiles of the original patient tumors including development. genomic mutational status, biochemical signaling, and response to tumor cell autonomously targeted therapeutics. PrimePanel provides Table 3: PDX-Derived Fresh Frozen Cells Validated in 3D Cell a versatile, cost-effective, high-throughput screening platform to as- Culture Assays sess drug efficacy prior to in vivo study. At CrownBio we have estab- lished five PrimePanel cell lines for 3D use, detailed in Table 4. PDX Model PDX Model Model No. Model No. Cancer Type Cancer Type Table 4: PrimePanel Cell Lines Validated in 3D Cell Culture Assays Bladder 2 Kidney 1 PrimePanel Cancer Type Patient and Disease Background ID Brain 2 Liver 11 CR5063CL Colon carcinoma Female aged 27. Breast 5 Lung 20 Prior treatment with FOLFOX, Camptosar®, Cervical 1 Lymphoma 3 Erbitux®, FUDR®, Leucovorin Cholangiocarcinoma 3 Metastatic Carcinoma 1 LU5143CL NSCLC Male, aged 78 Colorectal 18 Ovarian 6 LU5224CL SCLC Female, aged 48. Esophageal 7 Pancreatic 3 Invasive high grade carcinoma Gallbladder 2 Sarcoma 3 LU5381CL NSCLC Female, aged 57. Prior treatment with IFEX®, Taxol®, Gastric 10 Total 103 Gemzar® Head and Neck 5 ME5338CL Melanoma Metastatic melanoma (brain)

To complement our ex vivo models, CrownBio has established 3D cell cultures for >100 commercial cell lines that we have validated for in vitro drug efficacy studies, including breast, lung, ovary, and pancreatic cancer cell lines. A summary of our validatedin vitro 3D cell lines is shown in Table 5. Table 5: In Vitro Cell Lines Validated in 3D Cell Culture Assays

Methylcellulose Alvetex Soft Agar Cancer Type Cell Line Blood Daudi, DoHH-2, EHEB, Jurkat (clone E6-1), JVM-13, JVM-2, JVM-3, K-562, Karpas299, Kasumi, MEC-1, MEC-2, MEG-01, Molm-16, MOLT-4, Namalwa, OCI-LY19, REH, SU-DHL-10, SU-DHL-5, SUP-B15, TF-1, THP-1, Toledo, U-937, WSU-DLCL-2 Bone CADO-ES1, RD-ES Brain & Nerves A172, H4, IMR-32, LN18, LN229, SF126, SH-SY5Y, SK-N-SH, U-118 MG, U251, U-87 MG Breast BT-549, DU4475, MDA-MB-231, MDA-MB-436 MDA-MB-231 Colorectum HT-29, HCT 116, HT-29, HCT-116 Esophageal KYSE70, KYSE270, KYSE410, TE-1 Head & Neck FaDu, SW579 Liver HCCLM3, Hep G2, HLF, HUH-7, JHH-5, JHH-7, MHCC97H, PLC/PRF/5, SK-HEP-1, SNU-398, SNU-878, Hep 3B Hep 3B, Hep G2, JHH-7, SNU-886 PLC/PRF/5, SK-HEP-1 Lung A549, Calu-6, DMS53, DMS79, DMS114, EBC-1, H69AR, HCC4006, NCI-H1155, NCI-H1299, NCI-H1373, NCI-H1395, NCI-H1417, NCI-H1299 NCI-H1435, NCI-H1437, NCI-H157, NCI-H1581, NCI-H1650, NCI-H1688, NCI-H1703, NCI-H1792, NCI-H1836, NCI-H1975, NCI-H2052, NCI-H209, NCI-2227, NCI-H226, NCI-H23, NCI-H358, NCI-H446, NCI-H460, NCI-H520, NCI-H522, NCI-H526, NCI-H69, NCI-H82, SK-MES-1 Ovary OVCAR-8, SK-OV-3, SW626, SW756 Pancreas AsPc-1, Capan-1, MIAPaCa-2, PANC-1, PL45 CFPAC-1, MIAPaCa-2 Prostate PC-3 Stomach A3/KAW, AGS, AZ521, GTL-16, HGC-27, Hs 746T, IM95, IM95m, KATO III, MKN1, MKN45, MKN74, NCI-N87, NUGC-3, NUGC-4, AGS, AZ-521, GTL-16, OCUM-1, SCH, SNU-1, SNU-16, SNU-5, SNU-484, SNU601, SNU-620, SNU-638, SNU-668, SNU-719, YCC-2, YCC-7, YCC-10, YCC-11 HGC-27, MKN1

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Our 3D cell culture platforms utilizing the models detailed above for compound screening include the methylcellulose assay, including commer- cially available systems such as the 3D Alvetex® matrix (which can be used for cells which do not grow well in methylcellulose), soft agar colony formation, and the 3D tumor growth assay (3D TGA).

Ex Vivo Models in Methylcellulose Assays using HuPrime PDX Models Our 3D clonogenic in vitro/ex vivo methylcellulose assay provides an intimate connection between low cost, high throughput in vitro drug screen- ing, and clinically relevant in vivo drug efficacy studies with our unique HuPrime models (assay concept shown in Figure 1). All of the PDX-derived ex vivo models discussed above can be utilized in this assay.

Figure 1: Ex Vivo Clonogenic Assay Using Freshly Isolated Cells From HuPrime PDX Models

! ! Methylcellulose Format

Mid-Throughput Screen

Tissue Compound PDX Tumor Processing E cacy Test 3D Culture

Alvetex Format

For example, for freshly isolated primary cells inoculated into immunodeficient mice, xenografts are harvested once the tumor has reached the appropriate size (typically between 400mm3 and 800mm3). The harvested tumor is lysed and cells are seeded on methylcellulose or 3D Alvetex plates at the appropriate dilution to allow growth in 3D, with cell density optimized to provide highly reproducible results. The anticancer agent to be tested can then be administered to cells starting from Day 2 up to Day 10 of culture. Endpoints include phase contrast imaging of cells and CellTiter-Glo® (CTG) assay to verify anticancer compound efficacy.

An example ex vivo efficacy study for cisplatin on freshly isolated primary cells from the gallbladder tumor model GL0440 is shown inTable 6. Cispla- tin was administered to tumor cells at 0.1µM, 1µM, and 100µM and phase contrast images (10X) were taken for each dose to evaluate drug efficacy. Dose response curves can then be generated; Figure 2 shows the dose response to cisplatin for cells derived from the OV5397 ovarian cancer PDX model in the methylcellulose assay.

Table 6: Ex Vivo Efficacy Study of Cisplatin on Primary Cells Derived from GL0440 Grown in 3D TablTea 6bT:lae Eb x6lTe :Va E6bivx:l oe EV xE6iv f:Vof Ei cvExao fVcf Eiyicvf aSfoictc uEya fdcSfyitc uoSadtcfu yyC d oiSsyftp uoCldafis tyCpi niolsa fopt Cnilnai st Poipnrnli amo Ptnairn riPm yor Cnaimr ePylalr sCrimy eD Claelserr yilDvl sCe Derdil evlfser iodDvm efdr oGi vfmLreo0d Gm4 f4Lr G0o4 mLG40 r04Go 4LGw0rn 4oG 4iwrn0o n 3wG Dinr o 3iwnD n3 Din 3D CommentCommentComment [JB3]:Comment [JB3]: Ralph:[JB3]: Ralph: [JB3]: Ralph: Ralph: Vehicle controlVehicle controlVehicle controlVehicleVehicle control Control Cisplatin 0.1μMCisplatin 0.1μMCisplatinCisplatin 0.1μMCisplatin 0.1μM 0.1μM Cisplatin 1μMCisplatinCisplatin 1μMCisplatin 1μM 1μMCisplatin 1μM CisplatinCisplatin 100μMCisplatin 100μM 100μMCisplatin 100μMCisplatin 100μM hopefully these 4 images are hopefully these 4 images are hopefully these 4 images are hopefully these 4 images are ok to useok to useok to use ok to use

Figure Figure Figure 2: Dose Response Curve Figure 2: Dose Response Curve 2: Dose Response Curve 2: Dose Response Curve for Cfoisrfp oClaris tCpifnoilsa rEpt Cfilnafiest Eicpnftlf a Eoetfcnifnt e P ocErtnfi fmo ePncart riPm yor Cnaimr ePylalr sCrimy eD Claelserr yilDvl sCe Derdil evlfser iodDvm efdr oOi vfmrVeod5 Om 3fVr9 Oo57mV3 G95 rO73o 9VGw75rn 3oG 9iwrn7o n 3wG Dinr o 3iwnD n3 Din 3D CommentCommentComment [JB4]:Comment [JB4]: Ralph[JB4]: Ralph [JB4]: Ralph Ralph : have put excel file on : have put excel file on : have put excel file on : have put excel file on owncloud with the graph in,owncloud with the graph in,owncloud with the graph in,owncloud with the graph in, if you need it. if you need it. if you need it. if you need it. +1.855.827.6968 | [email protected] | www.crownbio.com

120 120120 120 Images are in an ppt file, let me know if they are ok as Images are in an ppt file, let me know if they are ok as Images are in an ppt file, let me know if they are ok as Images are in an ppt file, let me know if they are ok as 10 nM10 nM10 nM10 nM they are grouped or if you need different figsthey are grouped or if you need different figsthey are grouped or if you need different figsthey are grouped or if you need different figs 100 100100 100

80 80 80 80

60 60 60 60 1 µM1 µM1 µM 1 µM 40 40 40 40 Survival Rate (%) Survival Rate (%) Survival Rate (%) Survival Rate (%) 20 20 20 20

0 0 0 0 100 µM100 µM100 µM100 µM 0 0 020 020 2040 2040 4060 4060 6080 6080 8010080100100 100 Cisplatin (µM)Cisplatin (µM)Cisplatin (µM)Cisplatin (µM)

Ralph: Table 6: Ex Vivo Efficacy Study of Cisplatin on Primary Cells Derived from GL0440 Grown in 3D Comment [JB3]: Ralph:

Vehicle controlTable 6: Ex Vi vo Efficacy Study of CiCisplatin 0.1μMsplatin on Prim ary Cells Derived frCisplatin 1μMom GL0440 Grown in 3D Cisplatin 100μM Comment [JB3]: Ralph: hopefully these 4 images are ok to use Vehicle control Cisplatin 0.1μM Cisplatin 1μM Cisplatin 100μM hopefully these 4 images are ok to use 3D Cell Culture Platform In Vitro/Ex Vivo Assays for Cancer Pharmacology Factsheet

Figure 2: Dose Response Curve for Cisplatin Effect on Primary Ce lls Derived from OV5397 Grown i n 3D Comment [JB4]: Ralph Figure 2: Dose Response Curve Figure 2: Dose Response Curve for Cisplatin Effect on Primary Ex Vivo Models in 3D TGA Assays using HuPrimeComment [JB4]: Figure 2: Dose Response Curve Cells Derived from OV5397for Cis pGrownlatin E finfe c3Dt on Primary Cells Derived from OV5397 Grown in 3D Comment [JB4]: Ralph: have put excel file on PDX models and PrimePanel Cells owncloud with the graph in, if you need it. : have put excel file on 120 120 3D TGA assays have become a proven platform for oncology drug deowncloud with the graph in,Images are in an ppt file, let me know if they are ok as - if you need it. 120 10 nM Images are in an ppt file, let me know if they are ok as 10 nM velopment, with numerous publications covering research in a range they are grouped or if you need different figs 120100 they are grouped or if you need different figs 100 100 of cancer types(9,10,11,12). The 3D TGA assay utilizes a low stiffness lamiImages are in an ppt file, let me know if they are ok as -

) 10 10nM nM they are grouped or if you need different figs 80 % 10080 ( 80 nin rich extracellular matrix (IrBME, Cultrex®) to embed tumor cells, e t and admixing with hMSCs (e.g. IL-6, HGF) and CAFs provides the para- 60 a 60 80 R 60 l 1 µM crine signaling present in the TME of solid tumors. The addition of hor-

a 1 µM v 40 i 40 mones (e.g. DHT/E2), restriction of glucose (≤7mM), and maintenance 60 v

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Survival Rate (%) 1 µM Survival Rate (%) u of an acidic pH (6.8) provide a 3D assay that is both “humanized” and

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Survival Rate (%) TME-aligned for profiling of PDX-derived cells and drug panels . 0 100 µM100 µM 200 0 20 40 60 80 100 100 µM 0 20 40 Cispl60atin (µM)80 100 Table 7 shows a comparison between tumor conditions in humans, 0 20 40 60 80 100 100 µM 0 2D culture, and the humanized 3D cell culture environment, with Cisplatin (µM) 0 20 40 60 80 100 the 3D culture more closely representing the human condition. Cisplatin (µM) Table 7: Comparison of Tumor Conditions Across Humans, 2D, and 3D Culture(11) Our 3D TGA assay has been optimized to define a linear range for cell growth and for total cell population numbers using ala-

Tumor Condition Human 2D Culture 3D-TME Culture marBlue® colorimetric assay (further endpoints such as 3D Glo are also available). Individual cell types can also be monitored Oxygen (Hypoxia) 0-5% 21% ≤5% via fluorescent/bioluminescent read-outs if cells are transduc- Stiffness 200-4,000 Pa 3,000,000,000 Pa 200-4,000 Pa ed. The 3D TGA can be utilized with all of the ex vivo models de- pH acidic (≤7.0) buffered (≥7.2) acidic (≤7.0) scribed above for drug evaluation and further research as needed. Figure 3 shows example IC evaluations using our 3D TGA with Dimensionality 3D 2D 3D 50 selected colorectal, lung, and melanoma PrimePanel cell lines. Glucose availabilitty <7mM 10-25mM <7mM

Figure 3: Drug Panel IC50 Evaluation using PrimePanel Cell Lines in a 3D TGA

ME5338CL CR5063CL LU5224CL 1.5 1.5 Oxaliplatin 1.5 Etoposide Gemcitabine Carboplatin Cisplatin 5-FU Paclitaxel 5-FU Gemcitabine 1.0 1.0 Irinotecan 1.0 Paclitaxel U

U Etoposide Erbitux U Doxorubicin RF RF

RF Vinorelbine 0.5 0.5 0.5

-1 012345 -1 0 1 2 3 4 5 -1 0 1 2 3 4 5 Log [nM ] Log [nM] Log [nM ]

PrimePanel IC50 (µM) Cell Line 5-FU Carboplatin Cisplatin Doxorubicin Erbitux Etoposide Gemcitabine Irinotecan Oxaliplatin Paclitaxel Vinorelbine CR5063CL 0.405 - - - ~0.157 - 0.020 1.54 ~1.99 - - LU5224CL - ~0.014 - - - 0.248 0.080 - - ~0.017 0.021 ME5338CL 4.59 - >100 8.67 - ~2.09 - - - 0.007 -

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PDX tumor models are ideal for the preclinical assessment of anti- 3D TGA Large Scale Screens cancer agent efficacy because they closely mimic the pathological PDX models can be used in mouse clinical trials (HuTrials™) to identi- phenotypes of the original tumors. However, the labor-intensive, fy responder and non-responder populations, and to fully elucidate time-consuming, low throughput, and costly nature of in vivo animal signatures and predictive biomarkers for patient stratification in the studies can be limiting for their use in early stage drug development. clinic. Cells derived from these PDX can also be used in 3D TGA assays, PrimePanel cells are not generated from all PDX; however, the 3D TGA to perform large scale efficacy screens to potentially identify drugs system enables the use of fresh PDX material in a 96-well format. and combinations most likely to benefit individual patients(9). Correlation has been shown between response to anticancer agents in 3D TGA and in in vivo models and expected clinical outcome. In Vitro Models in Methylcellulose and Soft Agar LU6422 is a NSCLC PDX model of the adenocarcinoma subtype. The Assays using Cell Lines PDX model harbors a heterozygous c.2573T>G (L858R) in the intracel- To complement our ex vivo assays, CrownBio has validated a range of lular kinase domain, which is associated with hypersensitivity to EGFR commercially available in vitro cell lines for 3D assay. Growing cells are targeted therapies such as erlotinib in patients. In vivo, following sub- harvested and seeded on methylcellulose, soft agar, or other commer- cutaneous implantation with MSCs and treatment with 25 and 50mg/ cially available matrices at the appropriate dilution to achieve a final kg erlotinib, complete regression of all tumor growth is observed for cell density which is optimized for each cell line (e.g. 5,000 cells per this PDX model (Figure 4A). Cells derived from LU6422 are also hyper- well). Starting from Day 2, anticancer compounds are administered sensitive to erlotinib in 3D TGA, correlating with in vivo response (Fig- to each well according to desired concentration design. Cell growth ure 4B). These data were recently published with our collaborators in is monitored daily until endpoint, when cell number is counted using Molecular Cancer Therapeutics (9). CTG assay. Figure 5 shows a dose response curve for the head and neck cancer line FaDu, grown in 3D and treated with increasing doses of an experimental anticancer compound to test its efficacy. Figure 4: Correlation of LU6422 PDX Response In Vivo and in 3D TGA Figure 5: Dose Response Curve for the FaDu Head and Neck Cancer A. 1000 Cell Line Vehicle q.d. Erlotinib 25mg/kg q.d. 800 Erlotinib 50mg/kg q.d. 600 ) 3

500 DMSO Control 600

400 400 300 Tumor Volume (mm 200 200 Cell Viability (%T0) ↑ 100 TO 0 0 10 20 30 40 50 60 0 Study Day 0.01 0.1 1 10 100 1000 10000 100000 Compound XXX (nM) LU6422 B. 120 LU6422 + CAF 100 We also provide soft agar assays in low-attachment 96 well plates. Cell 3 80 lines are plated at 3×10 cells/well in 0.4% agar, over a base layer of 0.6% agar. The test agent is added with the growth media, and end- 60 points are CTG or imaging assays. Figure 6 shows comparison images 40 for both endpoints from a soft agar assay, for cisplatin dose response. Viability (%) The calculated EC values are comparable, validating imaging as an 20 50 endpoint for this assay. 0 0 0.01 0.1 1 10 100 [Erlotinib] (µM)

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Figure 6: Dose Response Curve for CFPAC-1 Cancer Cell Line Treated Our ex vivo and in vitro models can be utilized across a range of 3D with Cisplatin assays. CrownBio provides a 3D clonogenic in vitro/ex vivo methylcel- lulose assay which allows an intimate connection between low cost, high throughput in vitro drug screening and clinically relevant in vivo 120 CTG Assay 120 Imaging Assay drug efficacy studies. Soft agar assays provide a similar function, with 100 ) 100 ) %

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u 20 the TME with epithelial cells being cultured in combination with stro- 20 S S 0 0 mal cells and extra-cellular matrix in serum-free conditions, providing 0 20 40 60 80 100 0 20 a more40 clinically60 relevant80 culture100 method. In combination with PDX Cisplatin (µM) models,Cisplatin the (µM 3D) TGA has the potential to accelerate research by pro- viding a rapid and scalable ex vivo drug-sensitivity screen of multiple single and combinatorial agents(9).

C T G a s s Ca yT G a s s a y Im a g in g Iams sa ag yin g a s s a y 120 CTG Assay 120 Imaging Assay 1 0 0 1 0 0 1 0 0 1 0 0 CrownBio can be contacted at [email protected] for any fur- 100 9 0 9 0 ) 100 9 0 9 0 ) %

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60 l R l l l l 5 0 5 0 5 0 5 0 = 3.9µM l a a a a a v v v v a v i i i i 4 0 4 0 4 0 4 0 50 i v 40 v v 40 v v i r r r r v r u 3 0 u 3 0 u 3 0 u 3 0 v References EC r S S S S 2 0 2 0 u 2 0 2 0 u 20 20 S 1 S 1 0 1 0 1 0 1 0 Hutchinson L, Kirk R. High drug attrition rates – where are we going wrong? Nature Reviews Clinical On- 0 0 0 0 0 0 cology 2011;8: 189-190. 0 20 0 1 040 2 0 03 0 1 04 60 02 05 0 3 06 0 4 07 80 05 08 0 6 09 0 710100 0800 9 0 1 0 0 0 0 1 0 2200 03 0 1 04 0 2 04500 3 06 0 4 07 0 5 60800 6 09 0 7100 08800 9 0 1 0 0 100 C is p la tin ( CMis) p la tin ( M ) C is p la tin ( CMis) p la tin ( M ) 2Tian XF, Heng BC, Ge Z et al. Comparison of osteogenesis of human embryonic stem cells within 2D and 3D Cisplatin (µM) Cisplatin (µM) culture systems. Scandinavian Journal Clinical Laboratory Investigation 2008;68(1): 58-67. 3Elkayam T, Shaprut S, Dvir-Ginzberg M et al. Enhancing drug metabolism activities of C3A - A human hepatocyte cell line - By tissue engineering within scaffolds.Tissue Engineering 2006;12(5): 1357-1368 EC50 = EC503.7µM = 3.7µM EC50 = EC503.9µM = 3.9µM 4Shoeters G, Leppens H, Van Gorp U, and Van Den Heuvel R. Haemopoietic long-term bone marrow cultures from adult mice show osteogenic capacity in vitro on 3-dimensional collagen sponges. Cell Proliferation SummarSyu mmary 1992;25(6): 587-603. Summary 5Scaglione S, Braccini A, Wendt D et al. Engineering of osteoinductive grafts by expansion of ovine bone Tumors are 3D pathological entities. Their 3D nature should be taken into consideration Tumors are 3D pathological entities. Their 3D nature should be taken into consideration marrow stromal cells directly on 3D ceramic scaffolds. Biotechnology and Bioengineering 2005;93(1): 181- when assessing the efficacy of new anticancer agents when assessing the efficacy of new anticancer agents Tumors are 3D pathologicalas it is able to influence their response as it is able to influence their response entities. Their 3D nature should be tak- 187. to external stimuli such as . Attrition rateto external stimuli such as chemotherapy. Attrition rateen into considerations in oncology drug development whens in oncology drug development assessing the efficacyare are of new anticancer agents as it is able to influence their response to external stimuli such 6Debeb BG, Zhang X, Krishnamurthy S et al. Characterizing cancer cells with cancer stem cell-like features significantly highsignificantly higher than in other disease areas, with oer than in other disease areas, with only 5% of anticancer agentsnly 5% of anticancer agents which which in 293T human embryonic kidney cells. Molecular Cancer 2010;9: 180. show actshowivity in existing preclinic activity in existing preclinical moas chemotherapy.dels goal moingdels go on to be licensed after successful Phase III ing Attritionon to be licensed after successful Phase III rates in oncology drug development are (1) (1) 7Laurencin CT, El-Amin SF, Ibim SE et al. A highly porous 3-dimensional polyphosphazene polymer matrix trials . The majority of these failures are related to effictrials . The majority of these failures are related to efficsignificantly higheracy rather than toxicity issues, than acy rather than toxicity issues, in other disease areas, with only 5% of anti- for skeletal tissue regeneration. Journal Biomedical Materials Research 1996;30(2): 133-138. which confirmwhichs confirm that current preclinical oncologys that current preclinical oncologycancer agents 2D cell culture system which 2D cell culture system show activitys do not in existing srepresent do not rpreclinicalepresent, , models going 8Zhang Y, Ge Y, Liu Y et al. PrimePanel provides a high throughput in vitro drug screening platform that with sufficient fidelity, the corresponding 3D tumor with sufficient fidelity, the corresponding 3D tumor on to be licensedin situ after. in situ successful. Phase III trials(1). The majority of these intimately links to in vivo pharmacological analysis in PDX models [abstract]. In: Proceedings of the 104th failures are related to efficacy rather than toxicity issues, which con- Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Phila- CrownBio has developed a variety of CrownBio has developed a variety of ex vivo and ex vivoin vitro and models for 3D culture and in vitro models for 3D culture and delphia (PA): AACR; Cancer Research 2013;73(8 Suppl):Abstract nr 2787. preclinical oncology research. Our preclinical oncology research. Our ex vivofirms models are derived from our wellthatex vivo current models are derived from our well preclinical oncology-characterized 2D-characterized cell culture systems do not and validated and validated HuPrime Huand Prime HuKemia and represent,HuPDX models, and we provide freshly isolated cell lines, Kemia PDX models, and we provide freshly isolated cell lines, with sufficient fidelity, the corresponding 3D tumorin situ. 9Onion D, Argent RH, Reece-Smith AM et al. 3-Dimensional Patient-Derived Lung Cancer Assays Reveal Re- fresh frozen cells, as well as our proprietary fresh frozen cells, as well as our proprietary PrimePanelPrime cell lines which are early passage Panel cell lines which are early passage sistance to Standards-of-Care Promoted by Stromal Cells but Sensitivity to Histone Deacetylase Inhibitors. (<10) and maintain essential histopathological features and genetic profiles of the (<10) and maintain essential histopathological features and genetic profiles of the CrownBio has developed a variety of exoriginal vivo andoriginal in vitro models for Molecular Cancer Therapeutics 2016;15(4): 753-756. 10 patient tumors. To complement these resources, we also provide >100 commercial cell lines patient tumors. To complement these resources, we also provide >100 commercial cell lines 3D culture and preclinical oncology research. Our ex vivo models Casneuf T, Axel AE, King P et al. Interleukin-6 is a potential therapeutic target in interleukin-6 dependent, which we have also validated for 3D culture.which we have also validated for 3D culture.are derived from our well-characterized and validated HuPrime and estrogen receptor-α-positive breast cancer. Breast Cancer: Targets and Therapy 2016;8: 13–27. 11Krausz E, de Hoogt R, Gustin E et al. Translation of a Tumor Microenvironment Mimicking 3D Tumor Growth Our ex vivoOur and ex vivoin vitro and models can be utilized across a range of 3D assays. CrownBio in vitro models can be utilized across a range of 3D assays. CrownBio HuKemia PDX models, and we provide freshly isolated cell lines, fresh Co-culture Assay Platform to High-Content Screening. Journal of Biomolecular Screening 2013;18(1): 54-66. provides a 3D clonogenic provides a 3D clonogenic in vitro/ex vivoin vitrofrozen methylcellulose assay which provides an intimate /ex vivo cells, asmethylcellulose assay which provides an intimate well as our proprietary PrimePanel cell lines which are 12Spaeth EL, Dembinski JL, Sasser AK et al. Mesenchymal Stem Cell Transition to Tumor-Associated Fibro- connection between low cost, high throughput connection between low cost, high throughput early passagein vitro (<10) drug screening and clinically relevant in vitro and maintaindrug screening and clinically relevant essential histopathological features blasts Contributes to Fibrovascular Network Expansion and Tumor Progression. PLoS ONE 2009;4(4): 34992. in vivo drug efficacy studies. Soft agar assays provide a similar function, with CellTiterin vivo drug efficacy studies. Soft agar assays provide a similar function, with CellTiterand genetic profiles of the original patient-Glo or tumors.-Glo or To complement imaging endpoints as required.imaging endpoints as required. these resources, we also provide >100 commercial cell lines which we have validated for 3D culture. We also offer the 3D TGA, which haWe also offer the 3D TGA, which has become s become a proven platform for preclinical oncology drug a proven platform for preclinical oncology drug development. 3D TGA development. 3D TGA is closely aligned to the TME with epithelial cells being cultured in is closely aligned to the TME with epithelial cells being cultured in combination with stromal cells andcombination with stromal cells and extra-cellular matrix extra-cellular matrix in serumin serum-free conditions, providing -free conditions, providing Version 2.2 a more clinically relevant culture method. In combination with PDX models, the 3D TGA a more clinically relevant culture method. In combination with PDX models, the 3D TGA has has the potential tothe potential to accelerate research by providing a rapid accelerate research by providing a rapid and scalable and scalable ex vivo drugex viv-sensitivity o drug-sensitivity screen of mscreen of multiple singleultiple single and combinatorial agents and combinatorial agents(9). (9).

CrownBioCrownBio can be contacted at can be contacted at [email protected]@crownbio.com for any further questions or for any further questions or +1.855.827.6968 | [email protected] | www.crownbio.com information on our 3D cell culture platforms or for any other information on other information on our 3D cell culture platforms or for any other information on other CrownBioCrownBio products and services. products and services.